Early literature references to Rhizobium japonicum refer to strains characterized as "slow-growing" Rhizobia. More recent studies of biochemical and genetic characteristics have led to reclassification of "slow-growing" Rhizobia in the genus Bradyrhizobium (Jordan, D. C. (1982) Int. J. Syst. Bacteriol. 32:136. Furthermore, certain "fast-growing" strains have been found which are classified as R. japonicum on the basis of their ability to nodulate Glycine Max cv. Peking, an undeveloped Asian cultivar of soybean. Since the literature sometimes refers to slow-growing (Bradyrhizobium) strains simply as R. japonicum, confusion may occur. For clarity herein, "slow-growing" soybean nodulating strains are termed Bradyrhizobium japonicum strains, while "fast-growing" strains are termed Rhizobium japonicum strains. Similarly, Parasponia Rhizobium sp. has been reclassified as Bradyrhizobium sp. (Parasponia) (see, e.g. Scott, K. F. (1986), "Conserved nodulation genes from the non-legume symbiont Bradyrhizobium sp. (Parasponia), " Nucl. Acids Res. 14:2905-2919), and will be so referred to herein, although prior art references may specify the former name.
Biological nitrogen fixation in the root nodules of leguminous plants is a major component of world food production and therefore practical applications of this field are of major interest.
Prokaryotes can use a wide variety of nitrogen compounds as sole sources of cellular nitrogen. This variety includes ammonia, dinitrogen and nitrate among the inorganic compounds, and proline, arginine and glutamine among complex organic compounds. Each species can utilize a different array of nitrogen compounds. Glutamine, glutamate and aspartate are the key nitrogen compounds in intermediary metabolism. The latter two are the starting compounds of many pathways of amino acid biosynthesis and serve as amino group donors in many reactions. In all other cases the amino group is donated by glutamine. The major enzyme required for the assimilation of ammonia produced by N.sub.2 fixation is glutamine synthetase, which catalyses the reaction: EQU Glutamate+NH.sub.3 +ATP.fwdarw.glutamine+ADP+Pi.
At high NH.sub.4.sup.+ concentrations (&gt;1 mM) glutamate dehydrogenase is also found. Utilization of the assimilated ammonia depends on the activity of glutamate synthase catalyzing: EQU Glutamine+2-ketoglutarate+NADPH.fwdarw.2 glutamate+NADP.sup.+
Since ATP is hydrolysed, these reactions have a favorable equilibrium and allow the use of ammonia in the medium or ammonia derived enzymatically from other nitrogen sources (Meers, J. et al. (1970) J. Gen. Microbiol. 64:187-194). The formation of ammonia is thus a key step in the biological nitrogen cycle.
Biological nitrogen fixation can be achieved by a variety of microorganisms and occurs through the induction of an enzyme complex, nitrogenase, which converts atmospheric nitrogen to ammonia. This conversion occurs in a group of physiologically diverse prokaryotes, including facultative anaerobes (e.g., Klebsiella pneumoniae and Rhodosoirillum rubrum), obligate anaerobes (e.g., Clostridium pasteurianum), obligate aerobes (e.g., Azotobacter vinelandii) and some strains of blue-green algae (e.g., Anabaena cylindrica) (Sprent, J. I. (1979) The Biology of nitrogen fixing organisms, London, McGraw-Hill, pp. 8-11). While this enzyme complex is common to all characterized nitrogen fixing organisms, the conditions under which it is expressed vary considerably between species (Burns, R. C., Hardy, R. W. F. (1975): Nitrogen fixation in bacteria and higher plants, Springer-Verlag, Berlin). The first stages of nitrogen fixation, conversion of nitrogen into ammonia, are achieved symbiotically in the root nodules of leguminous plants which contain the nitrogen-fixing bacteria of the genera Rhizobium and Bradyrhizobium. Some non-leguminous plants, e.g., alder, also have interactions with symbiotic bacteria which are nitrogen fixers. In addition, free-living bacteria, e.g., Klebsiella pneumoniae and the photosynthetic blue-green bacteria, also fix nitrogen.
The symbiotic association between plants and bacteria of the genera Rhizobium and Bradyrhizobium is the result of a complex interaction between the bacterium and its host, requiring the expression of both bacterial and plant genes in a tightly coordinated manner (Vincent, J. M. (1980) In Symbiotic Association and Cyanobacteria, Nitrogen Fixation Vol. 2 (W. E. Newton, W. H. Orme-Johnson, eds.) Baltimore, University Park Press pp 103-129; and Verma, D. P. S. et al (1981) In Current Perspective in Nitrogen Fixation (A. H. Gibson, W. E. Newton, eds.) Canberra: Australian Academy of Science, pp. 205-208). In free-living rhizobial organisms nitrogenase synthesis is repressed and is only induced after the symbiotic relationship has been established. Furthermore, some species only interact with a narrow range of plant species, whereas other species interact with a wide range.
Bacteria bind to the emerging plant root hairs and invade the root tissue through the formation of an infection thread. The plant responds to this infection by the development of a highly differentiated root nodule. These nodules are the site of synthesis of the nitrogenase complex. Following nitrogen fixation, the fixed nitrogen is exported into the plant tissue and assimilated by the plant derived enzymes (Scott, D. B. et al. (1976) Nature 263:703,705).
Most rhizobial (this term includes Rhizobia and Bradyrhizobia) symbioses are confined to leguminous plants. Furthermore, strains which fix nitrogen in association with the agriculturally-important temperate legumes are usually restricted in their host range to a single legume genus. However, some rhizobial strains have been isolated which can fix nitrogen in a diverse group of legume species but can also form an effective symbiosis with non-legumes.
Despite the ability of certain plants to induce nitrogenase activity in a symbiotic relationship with some rhizobial species, the genetic analysis of biological nitrogen fixation has previously been confined to free living nitrogen fixing organisms, in particular Klebsiella pneumoniae. There are 17 linked nitrogen fixation (nif) genes arranged in at least 7 transcriptional units in the nif cluster of Klebsiella (Kennedy, C. et al. (1981) In Current Perspectives in Nitrogen Fixation (A. H. Gibson, W. E. Newton, eds.) Canberra: Australian Academy of Science, pp. 146-156; and Reidel et al. (1979) Proc. Nat. Acad. Sci. U.S.A. 76:2866-2870). Specific designations are assigned to nif genes, e.g. nifD, based on structural homologies to previously identified genes in other nitrogen fixing organisms at the DNA and protein levels. Three of the Klebsiella genes, nifH, nifD and nifK encode the structural proteins of the nitrogenase enzyme complex (viz. the Fe-protein subunit (dinitrogenase reductase) and the .alpha.- and .beta.-subunits of the Mo-Fe protein (dinitrogenase) respectively). Dinitrogenase is an .alpha.2.beta.2 tetramer in which the two non-identical .alpha. and .beta. subunits have similar molecular weights of 55,000 to 60,000. Dinitrogenase reductase is a dimer of two identical subunits each having a molecular weight around 35,000. These genes are linked in the same operon in K. pneumoniae and are transcribed from a promoter adjacent to the nifH gene. A similar situation (nifHDK) was found in two fast-growing rhizobia, R. meliloti (Ruvkun, G. B., et al. (1982) Cell 29:551-559) and R. leguminosarum (Schetgens, T. M. P. et al. (1984) "Identification and analysis of the expression of Rhizobium leguminosarum PRE symbiotic genes," p. 699, In C. Veeger and W. E. Newton (eds.) Advances in Nitrogen Fixation Research. Martinus Nijhoff/Dr. W. Junk Publishers, The Hague). In the slow-growing B. japonicum, it has been found that nifDK forms one operon and that nifH is located elsewhere on the genome (Fuhrmann, M. and H. Hennecke (1982) Mol. Gen. Genet. 187:419-425). A similar observation was made with another member of the slow-growing rhizobia, Bradyrhizobium sp. (Parasponia): a nifH region was found not to be linked to nifD (Scott, K. F., et al. (1983) DNA 2:141-148). Yet a different arrangement was detected in the cyanobacterium Anabaena sp. 7120, in which nifHD is separated from nifK (Rice, D., et at. (1982) J. Biol. Chem. 257:13157-13163). The remainder of symbiotic genes contain information required for bacterial attachment, root hair curling, initiation and development of nodules and establishment of symbiotic relationships. In addition, regulatory sequences such as promoters, operators, attenuators, and ribosome binding sites are found adjacent to the coding regions. These regulatory sequences control the expression of the structural genes, i.e., the coding sequences downstream in the 3'-direction of the DNA reading strand.
In rhizobia, nitrogenase synthesis is normally repressed under free-living conditions and is induced only within a complex symbiosis formed mostly with leguminous plants. R. trifolii is an example of a fast-growing Rhizobium with a narrow host range which cannot normally be induced to fix nitrogen in culture. In contrast, a Bradyrhizobium sp. (Parasponia) species has been isolated and this species is a slow-growing organism with a very broad host range capable of an effective symbiotic relationship with a broad variety of tropical legumes as well as the non-legume Parasponia (Ulmaceae) (Trinick, M. J. (1980) J. Appl. Bacteriol. 49:39-53). Bradyrhizobium sp. (Parasponia) can be induced to fix nitrogen in culture although the level of this fixation is about 100-fold less than can be obtained from the free-living bacterium Klebsiella pneumoniae. Other slow-growing rhizobia (Bradyrhizobia) include the commercially significant Bradyrhizobium japonicum, which nodulates soybeans.
The genetics of biological nitrogen fixation have been well characterized in the free-living organism Klebsiella pneumoniae. The structural genes for nitrogenase nifH, nifD and nifK encoding the Fe-protein subunit and the .alpha. and .beta. subunits of the Mo-Fe protein, respectively) have been mapped both genetically and physically (Kennedy, C. et al. (1981) In Current Perspectives in Nitrogen Fixation (eds. Gibson, A. H. and W. E. Newton) Australian Acad. Science, Canberra, pp. 146-156; and Reidel, G. E. et al. (1979), Proc. Nat. Acad. Sci. U.S.A. 76:2866-2870). Cloned DNA fragments carrying these sequences have been shown, by Southern blot analysis, to hybridize to homologous sequences in a wide range of nitrogen fixing organisms, including rhizobial species (Ruvkun, G. B. and F. M. Ausubel (1980) Proc. Nat. Acad. Sci. U.S.A. 77:191-195).
In spite of the ecological diversity of nitrogen fixing organisms, the physiological structure of the nitrogenase enzyme complex appears to be very conserved. In all cases where the enzyme complex has been purified, two proteins are present. The larger protein (dinitrogenase) contains molybdenum, iron and acid-labile sulfur, and carries the binding site for nitrogen and contains two subunit proteins .alpha.- and .beta.-coded by the nifD and genes respectively. The smaller protein (dinitrogenase reductase) contains iron and acid-labile sulfur, and is required for the reduction of the dinitrogenase and for the binding of MgATP used in this reduction. The dinitrogenase reductase is coded by the nifH gene. Chemical and spectral analyses of the purified protein components support a conservation of protein structure between organisms (Scott, K. F. et al. (1981) J. Mol. Appl. Genet. 1:71-81). In some cases the structures are sufficiently similar to allow formation of active hybrid enzymes between purified components, e.g., Azotobacter vinelandii and Klebsiella pneumoniae (Eady, R. R. and B. E. Smith (1979) In: A treatise on dinitrogen fixation I, II, eds. Hardy, R. W., Bottomley, F. and R. C. Burns, New York, Wiley Press pp. 399-490). Not surprisingly, therefore, the region of the nif operon coding for dinitrogenase reductase and dinitrogenase .alpha.-subunit and nifD) shows homology at the nucleic acid sequence level with the corresponding sequences in at least 19 other bacterial strains (Ruvkun, G. B. and F. M. Ausubel (1980) Proc. Nat. Acad. Sci. U.S.A. 77:191-195). Although this conservation of structure is generally true, significant differences between nitrogenases from different organisms also exist as can be shown by variable stability following purification and by the fact that active hybrid complexes do not form in all cases (Eady, R. R. and B. E. Smith (1979) supra).
A DNA fragment carrying the Klebsiella pneumoniae nifK, nifD and nifH genes has been isolated from the nif-strain UNF841(Tn5:nifK) (Cannon, F. C. et al. (1979) Mol. Gen. Genet. 174:59-66) and cloned into the Escherichia coli plasmid pBR325. The nucleotide sequences of the nifH gene and of 622 nucleotides of the nifD gene were determined (Sundaresan, V. and F. M. Ausubel (1981) J. Biol. Chem. 256:2808-2812; Scott, K. F. et al. (1981) supra). In addition, the DNA sequence of the nifH gene from Anabaena 7120 has been determined (Mevarech, M. et al. (1980) Proc. Nat. Acad. Sci. U.S.A. 77:6476-6480). A comparison of the two nucleotide sequences demonstrates two interesting features: (1) There is very little homology between the two sequences although a few stretches (up to 25 bp) are conserved, accounting for the observed interspecies homology of the nif genes (Ruvkun, G. B. and F. M. Ausubel (1980) supra); (2) In general, the promoter regions show very little sequence homology with the exception of a short region likely to be involved in common functions, e.g., RNA polymerase recognition.
In contrast, a comparison of the amino acid sequences of the dinitrogenase reductase and of the first 207 amino acids of the .alpha.-subunit of dinitrogenase of the two species and of another species show a much greater conservatism. The three species used in this comparison are Klebsiella pneumoniae (Kp); Anabaena 7120 (Ab); and Clostridium pasteurianum (Cp) (Tanaka, M. et al. (1977) J. Biol. Chem. 252:7093-7100). The Kp and Cp proteins share 67% amino acid sequence homology, Kp and Ab proteins share 71% homology, and the Cp and Ab proteins share 63%. This amino acid sequence homology is not spread evenly throughout the protein. Some regions are virtually identical (90% to 95% homology), while other regions are only weakly conserved (30-35% homology). The structural conservation appears to be centered around the five cysteine residues common to all three Fe proteins. These cysteine residues are believed to be ligands to the active center. Comparison of the N-terminal amino acid sequence of the .alpha.-subunit of dinitrogenase from Cp and Kp shows very little sequence homology in this region. This is in contrast to the very high conservation of amino acid sequence seen in the amino terminal region of the Fe protein. What little homology exists between Cp and Kp .alpha.-subunits is confined to regions around cysteine residues, as in the Fe proteins. These homologous regions are thought to be involved in the catalytic functions of the nitrogenase enzyme complex. Therefore, this structural conservatism is thought not to be the result of recent evolution and dispersal of the nif genes (Postgate, J. R. (1974) Sym. Soc. Gen. Microbiol. 24:263-292) but, rather, is postulated to be related to a conservation of function.
The discovery and study of plasmids, restriction enzymes, ligases and other enzymes involved in DNA synthesis has led to the rapidly developing field of genetic engineering. Use of these techniques has made it possible to transfer DNA across species boundaries, either from eukaryotic to prokaryotic organisms or vice versa. Alternatively, it has been possible to synthesize nucleotide sequences and to incorporate these synthetic sequences into living organisms where they have been expressed. For example, expression in E. coli has been obtained with DNA sequences coding for mouse dihydrofolate reductase (Chang, A. C. Y. et al. (1978), Nature 275:617-624) and for hepatitis B virus antigen (Burrell, C. J. et al. (1979), Nature 279:43-47). Two mammal hormones have also been produced in bacteria by use of synthetic DNA (Itakura, K. et al. (1977), Science 198:1056; and Goeddel, D. B. et al. (1979), Proc. Nat. Acad. Sci. U.S.A. 76:106). The practical application of DNA recombination requires the success of a number of different features. First, it must be possible to recognize the DNA fragment coding for the compound of interest and it must be possible to isolate that DNA fragment. Second, it is necessary to understand the mechanisms which control the expression of the information on that DNA fragment and to be able to transfer that information to the control of regulatory sequences which will maximize the productive capabilities of that information. This increased productive capacity could be by rearrangement of coding information and regulatory information within the same organism or between different organisms. The organisms involved may be prokaryotic or eukaryotic. Third, the conversion of coding information into useful products, such as storage proteins and hormones, must occur in an environment where they are not subsequently degraded.
In many cloning projects, only one of the two DNA strands is required initially. Many techniques have been used including poly(UG)-CsCl gradients (Szybalski, W. et al. (1971) Methods Enzymol., Grossman, L., and Moldave, K., eds. Vol.21D Academic Press, New York pp. 383-413), polyacrylamide gels (Maxam, A. and W. Gilbert (1977) Proc. Nat. Acad. Sci. U.S.A. 74:560-564), and exonuclease treatment (Smith, A. J. H. (1979) Nucl. Acids. Res. 6:831-848). An alternative biological approach has been developed using the bacteriophage M13. The replicative form of this phage DNA is a circular double stranded molecule; it can be isolated from infected cells and used to clone DNA fragments after which it can be reintroduced into Escherichia coli cells by transfection. M13 phage particles each containing a circular single stranded DNA molecule are extruded from infected cells. Large amounts of single stranded DNA containing a cloned fragment (5-110 .mu.g phage DNA/ml bacterial culture) can be easily and quickly recovered (Messing, J. et al. (1977), Proc. Nat. Acad. Sci. U.S.A. 74:3642-3646). The cloning of DNA fragments into the replicative form of M13 has been facilitated by a series of improvements which led initially to the M13mp7 cloning vehicle (Messing, J. et a). (1981), Nucleic Acids Res. 9:309-321). A fragment of the E. coli lac operon (the promoter and N-terminus of the .beta.-galactosidase gene) was inserted into the M13 genome. A small segment of DNA containing a number of restriction cleavage sites was synthesized and inserted into the structural region of the .beta.-galactosidase fragment. The M13mp7 DNA remains infective and the modified lac gene can still encode the synthesis of a functional .beta.-galactosidase .alpha.-peptide.
Following M13mp7, two new single stranded DNA bacteriophage vectors M13mp8 and M13mp9, have been constructed (Messing, J. and J. Vieira (1982) Gene 19:269-276). The nucleotide sequence of M13mp7 has been modified to contain only one each of the restriction sites (instead of two) and single restriction sites for HindIII, SmaI and XmaI have been added. Thus the restriction sites are EcoRI-SmaI-XmaI-BamHI-SalI-AccI-HincII-PstI-HindIII. These restriction sites have opposite orientations in M13mp8 and M13mp9. DNA fragments whose ends correspond to two of these restriction sites can be "force cloned" to one or the other of these two M13 cloning vehicles which have also been "cut" by the same pair of restriction enzymes. Thus a DNA fragment can be directly oriented by forced cloning. This procedure guarantees that each strand of the cloned fragment will become the (+) strand in one or the other of the clones and will be extruded as single stranded DNA in phage particles.
Restriction endonuclease cleavage fragments with non-complementing ends are seldom joined in a ligation. DNA cleaved by two different restriction endonucleases therefore cannot be circularized nor joined to another fragment produced by the same "two different restriction endonucleases" in both orientations. The result is that a recombinant molecule is formed during the ligation reaction with a defined order of the two fragments. Since the orientation of a cloned DNA fragment in the replicative form of M13 vectors determines which of the two DNA strands is going to be the viral strand, the use of M13mp8 or M13mp9 allows the direct preparation of one of the two DNA strands by cloning.